Time of Day
as a Conditioned Stimulus for the Effects of Nicotine, in Rats

Abstract

Research has shown that rats are
able to discriminate time of day as well as demonstrate environmentally
specific tolerance.It could then,
arguably, be assumed that rats could use time of day as a cue for predicting a
drug, such as nicotine.Based on this
premise, 7 Sprague-dawley rats were randomly assigned
to one of two squads.The first squad
(n=4), was injected with 0.4 mg/kg of nicotine bitartrate
at either 09:30 hrs. or17:30 hrs..The
second squad (n=3) was injected with the same dosage under the same conditions,
at 09:30 hrs. and17:30 hrs., but they were not assigned to always receive
it at one time or the other.On the test
day the dosage was increased to 0.6 mg/kg of nicotine.For squad one, the rat’s time of nicotine
injection was switched.The rats in
squad two’s dosage was also increased, and continued to be injected on the same
schedule.The results of this study
found that the rats that had been conditioned to a time of day had less of a
tolerance to the drug when the time of day was switched, than the rats who had gone through no conditioning.These results suggest that the rats in squad
one, during their conditioning trials associated time of day with receiving
nicotine, which is a conditioned compensatory response.These findings are valuable in a practical
sense because they can be applied to human smoking patterns.

Methods

Subjects

Eight adult, male Sprague-Dawley
rats, of varying weight, weighing between 370-496g were used in this study. The
eight rats were randomly placed in two squads, the first squad was made up of
four rats (n=4).The second squad was
made up of three rats (n=3).This study
was conducted under the schools animal care committee guidelines, and was umbrellaed under the animal lab.

Materials

The apparatus used to hold
the rats during sessions were individual
black cubes with no top, measuring 18x18x12.These cubes were videotaped by a small camera mounted on a movable
pole.The coding process of this study
involved a questionnaire, which is seen in the appendix, that requires the
observer to count the number of center movements and ataxic behavior that the
rat displays in fifteen minutes.They
are then asked to rate the intoxication level of each rat, based on these
observations.

Procedure

Conditioning took place over fourteen days.Each day the rats were injected twice, once
at 09:30 hrs. and once at 17:30 hrs.During
the morning conditioning session, each rat was removed from its cage, and
weighted on an electronic scale with a small metal basket.After being weighed the rat was returned to
its cage.Following the weighing of all
the rats, each rat was again individually removed from its cage and injected
with either 0.4 mg/kg of nicotine or saline.The first squad, the experimental group, received nicotine and saline at
specific times of day, two received nicotine in the morning and saline in the
afternoon, and two received saline in the morning and nicotine in the
afternoon. A control group, received nicotine and saline at specific times but
the drug received was random.The
experimental group was always injected and observed first and the control group
was always injected and observed second.Each rat was injected intraperitoneally (IP),
and was placed into his own individual black cube.The behaviors of the rats were then observed,
measured, and video taped for approximately fifteen minutes after each
injection.The behaviors that were measured
were ataxia, center-movement, and general intoxication level.In the testing session the exact same
procedure was used but the time of day that the drug was administered was
switched and the dose used was increased from 0.4 mg/kg to 0.6 mg/kg.

Results

Figure 1

Figure 1.
Level of intoxication as a function of

time of day
and dosage.In sessions 1 and 4 rat

received
nicotine in the AM.On test day, time of
day

was
switched, rat received nicotine in the PM, and

dosage was
increased to 0.6 mg/kg.

Figure 2

Figure 2.Level of intoxication as a function of

time of day
and dosage.In sessions 1 and 4 rat

received
nicotine in the AM.On test day, time of
day

was
switched, rat received nicotine in the PM, and

dosage was
increased to 0.6 mg/kg.

Figure 3

Figure 3.Level of intoxication as a function of time

of day and
dosage.In sessions 1 and 4 rat received

nicotine
in the PM.On test day, time of day was

switched,
rat received nicotine in the AM, and dosage

was
increased to 0.6 mg/kg.

Figure 4

Figure 4.Level of intoxication as a function of time

of day and
dosage.In sessions 1 and 4 rat received

nicotine in the PM.On test day, time of day was

switched,
rat received nicotine in the AM, and dosage

was
increased to 0.6 mg/kg.

Figure 5

Figure 5.Level of intoxication as a function of time

of day and
dosage.In sessions 1 and 4 rat received

nicotine in
either AM or PM.This was continued on

test day,
and dosage was increased to 0.6 mg/kg.

Figure 6

Figure 6.Level of intoxication as a function of time

of day
and dosage.In sessions 1 and 4 rat received

nicotine
in either AM or PM.This was continued
on

test
day, and dosage was increased to 0.6 mg/kg.

Figure 7

Figure 7.Level of intoxication as a function of time

of day and
dosage.In sessions 1 and 4 rat received

nicotine in
either AM or PM.This was continued on

test day, and dosage was increased
to 0.6 mg/kg.

Discussion

On a purely descriptive level these
results were in support of the hypothesis. It was shown that after tolerance
was obtained at a specific time of the day, a switch in the time of day when
the drug was presented, with a greater dose, caused the rat to revert back to
baseline or greater drug effects.This
would suggest that the rat had become conditioned to receiving the drug only at
the time of day that it was conditioned to.

In analyzing the results it is apparent that nicotine
had a much greater effect than saline on the rat’s intoxication levels, at both
times of day.The intoxication levels of
the rats dropped in the pretest trials because tolerance, as defined by Colman
(2001), to the drug was achieved.Therefore,
the effect of the nicotine decreased over time because the dose remained
unchanged throughout pretest sessions.At baseline the levels of intoxication were moderate to high.This was because the rats had never before
been exposed to the drug.Intoxication
levels in session 4 were lower because the rats had been exposed to the
nicotine and become tolerant (i.e. compensatory response) to its effects.Tolerance to a specific stimulus, time of
day, could not be shown until the test day, when the time of the drug was
switched.These results support the
literature on the effect of nicotine on rat’s activity levels, by Ksir (1994).In his
study he showed that relatively low dosed of nicotine cause
increased activity.He also showed that
doses higher than 0.4 mg/kg would decrease the activity level of the rat.It is important to acknowledge however, that
the level of activity in his study does not have the same implications as the
level of intoxication has in this study.

The change in the level of intoxication between
sessions 1 and 4 shows the tolerance that was achieved in this time.On the test day when the time of drug
administration was switched the rats showed an increase in drug effect.This was because during the conditioning
sessions, of rats 9 – 12, a conditioned compensatory response was elicited, to
the time of day. These findings are supported by Epstein, Caggiula,
and Stiller’s (1989) study, which explains that specific environments can be a
cue for the presence of a drug, in this study the environmental cue was time of
day.It is unlikely that any other
environmental cues caused the reappearance of the drug effect because all other
variables were held constant throughout all sessions.These findings were not only supported by the
literature but also by the results of the second squad of rats, 13-15.These results show that the rats that had not
been conditioned to receive nicotine at a specific time of day, did not have as
great of a drug effect on the test day, as the rats that were conditioned.This is because they had not conditioned a
compensatory response.The establishment
of this conditioned compensatory response is possible because, as seen in Mellgren, May, and Haddad (1983), rats have the ability to
discriminate time.Mean, Arolfo, Ginn and Pence (2000)
also contributed to this by showing that rats use time of day as an occasion
setter.These studies support the theory
that rats have an internal clock, which allows them to decipher when they are
receiving a drug.

Although this study does show that time of day can
act as a conditioned stimulus for the compensatory response to the nicotine,
some of the results do not support the findings of the study. These findings are included in the study to
ensure validity.These discrepancies are
likely to be due to human error.Although two different observers coded each session of videotape, to
ensure inter-observer reliability, differences in some of the observations may
have caused these findings.Another
factor that may have contributed to these discrepancies is the change of time
for day light savings.This occurred the
night before the test day.This was
however compensated for by injecting the rats and hour earlier, as though the
time change had not occurred.These
discrepancies are not strong enough to change the findings of the study but
should be accounted for because of their presence.

The
implications of this study have many practical applications.It suggested that these findings can be
applied to human behavior, and by finding that these rats associate time of day
with nicotine, it can be assumed that humans have the
same associative tendencies.What this
suggests is that humans may associate smoking cigarettes with a specific time
of day.It also addresses the issue of
cravings, the feeling of need, that people tend to have when addicted to
cigarettes.For example, an individual
may always smoke a cigarette in the morning when they wake up.Then one morning they do not smoke, they will
crave the cigarette. This is because of their body’s conditioned compensatory
response to the nicotine in the morning.Their body is conditioned to receiving nicotine every morning so it
prepares itself for the presence of the drug, and when it is not presented the
body has over compensated which causes cravings.Knowledge of information such as this can
help in preventing drug and nicotine use in humans.By knowing when they will be more attracted
to the drug, health professionals can better aid them in overcoming their addictions.